1. Field of the Invention
The present invention relates generally to the fields of molecular separation and particle discrimination. More particularly, it concerns the fractionation of particulate matter utilizing a combination of electrical, hydrodynamic or gravitational forces.
2. Description of the Related Art
The ability to identify, characterize and purify cell subpopulations is fundamental to numerous biological and medical applications, often forming the starting point for research protocols and the basis for current and emerging clinical protocols. Cell separation has numerous applications in areas such as medicine, biotechnology, biomedical research, environmental monitoring and bio/chemical warfare defense. For example, cell separation can make possible life-saving procedures such as autologous bone marrow transplantation for the remediation of advanced cancers where the removal of cancer-causing metastatic cells from a patient""s marrow is necessitated (Fischer, 1993). In other applications, such as the study of signaling between blood cells (Stout, 1993; Cantrell et al., 1992), highly purified cell subpopulations permit studies that would otherwise be impossible. Current approaches to cell sorting most frequently exploit differences in cell density (Boyum, 1974), specific immunologic targets (Smeland et al., 1992), or receptor-ligand interactions (Chess et al., 1976) to isolate particular cells.
These techniques are often inadequate and sorting devices capable of identifying and selectively manipulating cells through novel physical properties are therefore desirable. The application of the principles of AC electrokinetics has been used for the dielectric characterization of mammalian cells through the method of electrorotation (ROT) (Arnold and Zimmermann, 1982; Fuhr, 1985; Hxc3x6lzel and Lamprecht, 1992; Wang et al., 1994) and for cell discrimination and sorting (Hagedorn et al., 1992; Huang et al., 1993; Gascoyne et al., 1992; Gascoyne et al., 1994; Huang et al., 1992). In these techniques, cells become electrically polarized when they are subjected to an AC electric field. In ROT, a rotational electrical field is applied and the interaction between the cells"" polarization and the applied field results in cell rotation. If that field is inhomogeneous, then the cells experience a lateral dielectrophoretic (DEP) force, the frequency response of which is a function of their intrinsic electrical properties (Gascoyne et al., 1992). In turn, these properties depend strongly on cell composition and organization, features that reflect cell morphology and phenotype. Cells differing in their electrical polarizabilities can thus experience differential forces in the inhomogeneous electric field (Becker et al., 1994; Becker et al., 1995). Analysis of the dielectrophoretic motion of mammalian cells as a function of applied frequency permits cell membrane biophysical parameters, such as capacitance and surface conductance, to be probed. Because DEP effectively maps biophysical properties into a translational force whose direction and magnitude reflects cellular properties, DEP force may induce separation between particles of different characteristics. For example, DEP has been used on a microscopic scale to separate bacteria from erythrocytes (Markx et al., 1994), viable from nonviable yeast cells (Wang et al., 1993), and erythroleukemia cells from erythrocytes (Huang et al., 1992). However, the differences in the electrical polarizabilities of the cell types in those various mixtures were greater than those to be expected in many typical cell sorting applications.
Field flow fractionation (FFF) has also been generally employed for separation of matter, utilizing particle density, size, volume, diffusivity, and surface charge as parameters (Giddings, 1993). The technique can be used to separate many different types of matter, from a size of about 1 nm to more than about 100 micrometers, which may include, for example, biological and non-biological matter. Separation according to field flow fractionation occurs by differential retention in a stream of liquid flowing through a thin channel. The FFF technique combines elements of chromatography, electrophoresis, and ultracentrifugation, and it utilizes a flow velocity profile established in the thin channel when the fluid is caused to flow through the chamber. Such velocity profile may be, for example, linear or parabolic. A field is then applied at right angles to the flow and serves to drive the matter into different displacements within the flow velocity profile. The matter being displaced at different positions within the velocity profile will be carried with the fluid flow through the chamber at differing velocities. Fields may be based on sedimentation, crossflow, temperature gradient, centrifugal forces, and the like. The technique suffers, however, from producing insufficiently pure cell populations, being too slow, or being too limited in the spectrum of target cells or other matter.
Thus, there exists a need in the art for highly discriminate separation of particulate matter, especially biological matter. Furthermore, such a technique should operate without physically modifying the structure of the matter to be separated. In addition, it should allow for the sensitive manipulation of such particles, which may include characterization and purification of desired matter from extraneous or undesired matter.
The present invention seeks to overcome these drawbacks inherent in the prior art by combining the use of frequency-dependent dielectric and conductive properties of particles with the properties of the suspending and transporting medium. As used herein, the term xe2x80x9cmatterxe2x80x9d is intended to include particulate matter, solubilized matter, or any combination thereof. The invention provides a novel apparatus and novel methods by which different particulate matter and solubilized matter may be identified and selectively manipulated. These particles may also be fractionated or collected separately by changing the DEP force or the fluid flow characteristics. Utilizing the invention in this manner, particulate matter and solubilized matter may be discriminated and separated. The apparatus and methods of the present invention may discriminate many different types of matter simultaneously.
The present invention provides a method and apparatus for the discrimination of particulate matter and solubilized matter of different types. This discrimination may include, for example, separation, characterization, differentiation and manipulation of the particulate matter. According to the present invention, the particulate matter may be placed in liquid suspension before input into the apparatus. The discrimination occurs in the apparatus, which may be a thin, enclosed chamber. Particles may be distinguished, for example, by differences in their density, size, dielectric permittivity, electrical conductivity, surface charge, and/or surface configuration. In the case of the biological cells, they may be discriminated according to differences in their size, density, membrane electrical capacitance and conductance, interior conductivity and permittivity, and/or surface charges.
The methods according to the present invention may be used to discriminate particulate matter, including inorganic matter, such as minerals, crystals, colloidal, conductive, semiconductive or insulating particles and gas bubbles. The methods of the present invention may also be used to discriminate biological matter, such as cells, cell organelles, cell aggregates, nucleic acids, bacterium, protozoans, or viruses. Further, the particulate matter may be, for example, a mixture of cell types, such as fetal nucleated red blood cells in a mixture of maternal blood, cancer cells such as breast cancer cells in a mixture with normal cells, or red blood cells infested with malarial parasites. Additionally, the methods of the present invention may be used to discriminate solubilized matter such as a molecule, or molecular aggregate, for example, proteins, or nucleic acids.
Particles to be discriminated may be any size. However, the present invention is generally practical for particles between xcx9c10 nm and xcx9c1 mm, and may include, for example, chemical or biological molecules (including proteins, DNA and RNA), assemblages of molecules, viruses, plasmids, bacteria, cells or cell aggregates, protozoans, embryos or other small organisms, as well as non-biological molecules, assemblages thereof, minerals, crystals, colloidal, conductive, semiconductive or insulating particles and gas bubbles. For biological applications using living cells, the present invention allows cells to be separated without the need to alter them with ligands, stains, antibodies or other means. Cells remain undamaged, unaltered and viable during and following separation. Non-biological applications similarly require no such alteration. It is recognized however, that the apparatus and methods according to the present invention are equally suitable for separating such biological matter even if they have been so altered.
The apparatus may include, for example, a chamber. The chamber may have at least one inlet and one outlet port, an interior surface and an exterior surface. The chamber may further be designed to have structural characteristics so that a desired flow velocity profile is generated when a fluid is caused to flow through the chamber. The flow velocity profile refers to the fact that the fluid at different positions travels at differing velocities. The chamber may be rectangular in shape and may include, for example, a top wall, bottom wall and two side walls. In certain embodiments, the chamber may be constructed so that the top wall and bottom wall are of a much greater magnitude than the side walls, thereby creating a thin chamber capable of creating a velocity profile. For example, for a rectangular chamber having a width W, a height H and a length L where the condition W greater than  greater than H is held, the velocity profile for a fluid flow in the chamber in the direction along its length can be described as follows (Pazourek and Chmelik, 1992),                               V          ⁡                      (                          x              ,              h                        )                          =                  6          ⁢                      (                          V              m                        )                    ⁢                      h            H                    ⁢                      (                          1              -                              h                H                                      )                    ⁢                      (                          1              -                                                cosh                  (                                      2                    ⁢                                          3                                        ⁢                                                                  (                                                  x                          -                                                      W                            /                            2                                                                          )                                            /                      H                                                                                        cosh                  ⁡                                      (                                                                  3                                            ⁢                                              W                        /                        H                                                              )                                                                        )                                              (        1        )            
where x is the distance from the chamber side wall (measuring horizontally) and h is the distance from the bottom wall (measuring vertically). The factor  less than Vm greater than  is the average velocity of the fluid traveling through the chamber. Equation (1) indicates that the fluid velocity follows a parabolic dependency on the vertical position in the chamber. Since W greater than  greater than H, the fluid velocity expression in Equation (1) can be approximated as a parabolic dependency, as                                           V            ⁡                          (              h              )                                =                      6            ⁢                          (                              V                m                            )                        ⁢                          h              H                        ⁢                          (                              1                -                                  h                  H                                            )                                      ,                            (        2        )            
where the edge-effect along the chamber width is ignored. In such a flow profile, the fluid velocity increases with increasing distance from the chamber top or bottom walls. Fluid close to the top and bottom walls travels at near zero velocity; and that at the mid point between the top and bottom walls travels at the highest velocity.
In other embodiments, the chamber may be constructed so that the top wall and bottom wall are of a much smaller magnitude than the side walls, again creating a thin chamber capable of creating a velocity profile. In that case, the parameter h in equation (2) describes the horizontal distance in the chamber from a side wall and H is the width of the chamber. Alternately, the chamber may be of circular construction, triangular, rectangular, hexadecagonal, or of other geometrical shapes. In such cases, modified versions of equation (2) will apply as is known in the art of hydrodynamics. As such, the present invention is not intended to be limited to a particular geometric shape. The chamber according to the present invention may be constructed of many different materials, for example, glass, polymeric material, plastics, quartz, coated metal, or the like.
The chamber includes at least one electrode element adapted along a portion or all of the chamber walls. Each of these one or more electrode elements may be electrically connected to an electrical conductor, which in turn are connected to an electrical signal source. In the discussion which follows, the terms xe2x80x9celectrode elementxe2x80x9d or xe2x80x9celectrodesxe2x80x9d will be used. As used herein, xe2x80x9celectrode elementxe2x80x9d is a structure of highly electrically-conductive material over which an applied electrical signal voltage is constant. It is to be understood that these terms include all of the electrode configurations described below. An electrical signal generator, which may be capable of varying voltage, frequency, phase or all the three may provide at least one electrical signal to the electrode elements. The electrode elements of the present invention may include, for example, a plurality of electrode elements which may be connected to a plurality of electrical conductors, which in turn are connected to the electric signal generator.
The chamber according to the present invention may include a plurality of electrode elements which comprise an electrode array. As used herein, an xe2x80x9celectrode arrayxe2x80x9d is a collection of more than one electrode element in which each individual element may be displaced in a well-defined geometrical relationship with respect to one another. This array may be, for example, an interdigitated (or parallel) array, interdigitated castellated array, a polynomial array, plane electrode, or the like. Further, the array may be comprised of microelectrodes of a given size and shape, such as an interdigitated array. The electrode array may be adapted along any interior or exterior surface of the chamber. Alternately, it is envisioned that the electrode array may be incorporated into the material which comprises the chamber walls. In certain embodiments, the electrode array may be a multilayer array in which conducting layers may be interspersed between insulating layers. Further, the present invention may have a plurality of electrode arrays which may be adapted, for example, on opposing surfaces of the chamber. However, it may be possible to place the plurality of electrode arrays on adjacent surfaces or on all surfaces of the chamber. Fabrication of such an electrode array, depending on electrode dimensions, may use any of the standard techniques known in the art for patterning and manufacturing microscale structures.
For an interdigitated (parallel) array, the parallel electrode elements may be adapted to be substantially longitudinally or latitudinally along a portion of the chamber. Other configurations of electrode elements are contemplated by the present invention, such as electrode elements adapted at angles to the chamber. It is also possible to use a three-dimensional electrode element that may or may not be attached to the surface of the chamber. For example, electrode elements may be fabricated from silicon wafers, using the semiconductor microfabrication techniques known in the art. If the electrodes are adapted along the exterior surface of the chamber, it is envisioned that a means of transmitting energy into the chamber, such as a microwave transmitter may be present. The electrode elements may be configured to be on a plane substantially normal or parallel to a flow of fluid travelling through said chamber. However, it is to be understood that the electrode elements may be configured at many different planes and angles to achieve the benefits of the present invention.
When the electrode elements are energized by at least one electrical signal from the electrical signal generator, the electrode elements thereby create spatially inhomogeneous alternating electric field, which causes a DEP force on the particulate matter and solubilized matter. This DEP force may be a conventional DEP force (cDEP), or it may be a travelling wave DEP force (twDEP), or the combination of the both. The cDEP force may have components acting in a direction substantially normal to the electrode element plane, that is, the cDEP force may cause matter to move towards or away from this plane. In reference to the fluid traveling through the chamber, the DEP force may act substantially in a direction normal to the fluid flow. As used herein, xe2x80x9ca direction normal to the fluid flowxe2x80x9d means in a direction which is substantially non-opposing and substantially nonlinear to the flow of a fluid traveling through the chamber. This direction may be for example, vertically, sideways, or in another non-opposing direction. By effect of this DEP force, particulate matter and solubilized matter is displaced to different positions within the fluid, in particular within the flow velocity profile established in the chamber. This displacement may be relative to the electrode elements, or may relate to other references, such as the chamber walls.
It is noted that by altering phase of the alternating electrical signal, a second DEP force, known as traveling wave DEP (twDEP), is created. The cDEP force is dependent on the spatial inhomogeneity of the electric field and causes matter to move towards or away from regions of high electrical field strength. The twDEP force is dependent upon the phase distribution of the applied electric field, and caused matter to move towards or away from the direction of increasing phase values.
In the present invention, the cDEP force is dependent on the magnitude of the spatial inhomogeneity of the electric field and the in-phase (real) part of the electrical polarization induced in matter by the field. It is to be understood that the term xe2x80x9celectrical polarizationxe2x80x9d is related to the well known Clausius-Mossotti factor, described below. This field-induced electrical polarization is dependent on the differences between the dielectric properties between the matter and the suspending medium. These dielectric properties include dielectric permittivity and electrical conductivity. Together, these two properties are known as complex permittivity. The cDEP force causes the matter to move towards or away from regions of high electrical field strength, which in an exemplary embodiment, may be towards or away from the electrode plane.
The equation for the time-averaged dielectrophoretic force in a non-uniform electrical field is (Wang et al., 1995):
FDEP=2xcfx80∈mr3Re(fCM)∇Erms2+2xcfx80∈mr3Im(fCM) (Ex02∇xcfx86x+Ey02∇xcfx86y+Ez02∇xcfx86z)xe2x80x83xe2x80x83(3)
where Erms is the rms value of the electric field distribution, Excex10 and xcfx86xcex1 (xcex1=x,y,z) are the magnitudes and phases of each field component. The parameters Erms, Excex10 and xcfx8660  are, in general, functions of spatial coordinates (x,y,z) and dependent on positions. Nevertheless for the sake of simplicity, the explicit spatial coordinates (x,y,z) have be omitted. The factor fCM is the well-known Clausius-Mossotti factor, defined as             f      CM        =                  (                              ϵ            p            *                    -                      ϵ            m            *                          )            /              (                              ϵ            p            *                    -                      2            ⁢                          xe2x80x83                        ⁢                          ϵ              m              *                                      )              ,
where ∈*p and ∈*m are the complex permittivities of the matter and its suspending medium, respectively. Each complex permittivity is defined as             ϵ      *        =          ϵ      -                        i          σ                /                  (                      2            ⁢                          xe2x80x83                        ⁢            π            ⁢                          xe2x80x83                        ⁢            f                    )                      ,
∈ and "sgr" are the permittivity and conductivity, respectively. The parameters is the frequency of the applied field, r is the radius of the matter on which the DEP force is acting. Equation (3) indicates that dielectrophoretic force in general consists of two components. The first component, cDEP (conventional dielectrophoretic) force component, is dependent on the real part Re(fCM) (in-phase component) of the Clausius-Mossotti factor fCM and the magnitude non-uniformity factor ∇Erms2 of the applied electric field. If the in-phase part of the Clausius-Mossotti factor is greater than zero, then the cDEP force component will move the matter towards the location of the strong field. If the in-phase part of the Clausius-Mossotti factor is less than zero, the cDEP force component will move the matter towards the location of the weak field. The second component, twDEP (traveling-wave dielectrophoretic) force component, is dependent on the imaginary part Im(fCM) (out-of-phase component) of the Clausius-Mossotti factor fCM and the phase non-uniformity factor (∇xcfx86x, ∇xcfx86y and ∇xcfx86z) of the applied electric field. Depending on the polarity of Im(fCM), the twDEP force component will tend to move the matter towards the direction of increasing or decreasing phase values (xcfx86x,xcfx86y,xcfx86z) of the field components. Whether the matter experiences a cDEP force component, or a twDEP force component, or both, will depend upon the electrode geometry and the manner in which the electrical signals are applied.
In the present invention, the purpose of applying the DEP force is to cause particulate matter and solubilized matter to be displaced to different positions within the fluid flow velocity profile established in the chamber. Specifically, the DEP force is applied so that it acts in conjunction with other forces so that different types of particulate matter is equilibrated at different, characteristic positions within the flow profile (or solubilized matter will attain equilibrium concentration distribution within the flow profile). Examples of other forces that may be used in conjunction with the applied DEP force include gravitational forces, electrical forces and hydrodynamic lifting forces. Gravitational forces arise because of the density difference between the matter and its suspending medium. If the density of the matter is larger than that of the medium, the matter will experience a gravitational force pointing downwards. If the density of the matter is smaller than that of the medium, the matter will experience a gravitational force pointing upwards. An electrical force may be produced on the charged matter when a DC electrical field is established in the chamber. Hydrodynamic lifting forces refer to the forces acting on matter when it is close to a chamber wall and there is a fluid-velocity profile in the chamber (Williams et al., 1992). Such lifting forces tend to push the matter away from the chamber walls. The magnitude of the hydrodynamic lifting forces may depend on the size, density, shape of the matter, the density and viscosity of the medium, and the fluid-flow profile in the chamber. In some cases, the hydrodynamic lifting forces may be significant and may be of comparable magnitude to the gravitational forces and dielectrophoretic forces. In other cases, the hydrodynamic lifting forces may be much smaller than the dielectrophoretic forces and may play a negligible role in positioning matters in the hydrodynamic flow profile.
A feature of the present invention is that DEP forces are applied to the matter in combination with at least one other force so that matter having different properties (dielectric/electrical property, density property, charge property) will be positioned differently in the flow-velocity profile established in the chamber. In one embodiment, DEP forces may be balanced with gravitational forces so that different matter attains different equilibrium positions. In another embodiment, DEP forces may be balanced with gravitational forces plus the hydrodynamic lifting forces so as to influence the equilibrium positions of the matter. In another embodiment, DEP forces may be balanced by electrical forces to control the equilibrium positions of different matter. DEP and other forces depend on the properties of the matter (e.g. dielectric property, density, size, electrical charge etc), therefore, the balance of these forces and the resulting matter equilibrium positions (or displacement) are also dependent on the properties of the matter. The matter of different properties will be displaced to different positions within the chamber or within the flow-velocity profile. These equilibrium positions may also be referred as xe2x80x9clevitationxe2x80x9d or xe2x80x9clevitation heightxe2x80x9d. As used herein, xe2x80x9clevitatexe2x80x9d or xe2x80x9clevitation heightxe2x80x9d means that matter is displaced at different levels with respect to the electrode elements, in any direction, or matter is equilibrated at different positions with respect to the electrode elements under the balance of DEP forces and other forces.
In one embodiment of the present invention, an interdigitated (or parallel) electrode array may be adopted on the bottom wall of the separation chamber. The geometry of the interdigitated electrode array is characterized by the electrode element width to electrode element spacing. In one embodiment, the electrode element width and spacing are the same and an electrical voltage is applied to the neighboring electrode elements. Under this condition, only the cDEP force component is present in DEP forces exerting on the matter in the chamber. This cDEP force mainly lies in the vertical direction, especially for positions some distances away from the chamber bottom wall. This force acting on a matter of the radius r can be approximated as (Huang et al., 1997; Wang et al., 1998).
FDEP=2xcfx80∈mr3Re(fCM)U2A exp(xe2x88x922xcfx80h/d)xe2x80x83xe2x80x83(4)
where U is the applied root-mean-squared (RMS) voltage, ∈m is the dielectric permittivity of the medium. The DEP forces fall approximately exponentially with height h above the electrode plane, with a decay constant that is characterized by the periodic distance d(=2*electrode-element-width+2*electrode-element-spacing) of the electrode array and a unit voltage force coefficient A. Thus, changing electrode element width and/or spacing may modify DEP forces acting on the matter. The DEP forces shown Equation (4) may be used to balance the gravitational forces acting on the matter to achieve positioning particulate matter at different heights from the electrode plane. The gravitation forces are given by xe2x88x924/3xcfx80r3(xcfx81pxe2x88x92xcfx81m). Here xcfx81p and xcfx81m are the densities of the matter and its suspending medium, respectively, satisfying the relationship xcfx81p greater than xcfx81m. The balance of gravitational and DEP levitation forces positions the matter at a stable equilibrium height, given by                               h          eq                =                              d                          4              ⁢                              xe2x80x83                            ⁢              π                                ⁢                                    ln              ⁡                              (                                                                            3                      ⁢                                              xe2x80x83                                            ⁢                                              ϵ                        m                                            ⁢                      U                                                              2                      ⁢                      g                                                        ⁢                                                            A                      ⁢                                              xe2x80x83                                            ⁢                                              Re                        ⁡                                                  (                                                      f                            CM                                                    )                                                                                                            (                                                                        ρ                          p                                                -                                                  ρ                          m                                                                    )                                                                      )                                      .                                              (        5        )            
Thus, equilibrium levitation heights are dependent on the dielectric property (as characterized by the dielectric polarization factor, Re(fCM)) and density (xcfx81p) of the matter, of the electrode dimensions (A and d), of the applied DEP field strength (U). The factor Re(fCM) is also dependent on the frequency of the applied field. In this embodiment, the DEP force acts in combination with the gravitational forces, and the levitation height of the matter is in the vertical direction with respect to the electrode plane. In other embodiments, the DEP force may act in combination with other forces and the levitation height may not be along the vertical direction.
In another embodiment, the interdigitated electrode array may have different electrode width from electrode spacing. The DEP force may take different form from those shown in Equation (4). Thus, the ratio of electrode width to electrode spacing may be modified to change the particulate matter and solubilized matter levitation height. Specifically, by changing this ratio, the electric field which is created is thereby altered. When the electric field is thereby altered, in magnitude and/or inhomogeneity, the levitation height of the matter similarly change. This levitation need not be in a vertical direction, and may include displacement in a horizontal direction, for example.
Common electrical conductors may be used to connect the one or more sets of electrode elements to the signal generator. The common electrical conductors may be fabricated by the same process as the electrodes, or may be one or more conducting assemblies, such as a ribbon conductor, metallized ribbon or metallized plastic. A microwave assembly may also be used to transmit signals to the electrode elements from the signal generator. All of the electrode elements may be connected so as to receive the same signal from the generator. It is envisioned that such a configuration may require presence of a ground plane. More typically, alternating electrodes along an array may be connected so as to receive different signals from the generator. The electrical generator may be capable of generating signals of varying voltage, frequency and phase and may be, for example, a function generator, such as a Hewlett Packard generator Model No. 8116A. Signals desired for the methods of the present invention are in the range of about 0 to about 50 volts, and about 0.1 kHz to about 180 MHz, and more preferably between about 0 to about 15 volts, and about 10 kHz to 10 MHz. These frequencies are exemplary only, as the frequency required for matter discrimination is dependent upon the conductivity of for example, the cell suspension medium. Further, the desired frequency is dependent upon the characteristics of the matter to be discriminated. The variation of the frequency will generally alter the polarization factor (the Clausius-Mossotti factor) of the matter and change the DEP forces exerted on the matter. Thus to enhance the discrimination of matters using the present invention, the operational frequency may be chosen so as to maximize the difference in the DEP forces exerting on the matter or maximize the difference in the DEP force induced levitation height between different matter. In one embodiment of the invention using the interdigitated (parallel) electrode array, the levitation-height of the matter may be expressed in Equation (5). As an example, the operation frequency for discriminating two different matters (A and B) with such an embodiment of the invention may be chosen to maximize the levitation height difference |heqAxe2x88x92heqB|:                               Max          frequency                ⁢                  "LeftBracketingBar"                                    h              eqA                        -                          h              eqB                                "RightBracketingBar"                ⁢                  xe2x80x83                ⁢        or                            (        6        )                                          Max          frequency                ⁢                              "LeftBracketingBar"                                          d                                  4                  ⁢                                      xe2x80x83                                    ⁢                  π                                            ⁢                              ln                ⁡                                  (                                                                                    (                                                                              ρ                            pB                                                    -                                                      ρ                            m                                                                          )                                            ⁢                                              Re                        ⁡                                                  (                                                      f                            CMA                                                    )                                                                                                                                    (                                                                              ρ                                                          p                              ⁢                                                              xe2x80x83                                                            ⁢                              A                                                                                -                                                      ρ                            m                                                                          )                                            ⁢                                              Re                        ⁡                                                  (                                                      f                            CMB                                                    )                                                                                                      )                                                      "RightBracketingBar"                    .                                    (        7        )            
Here the polarization factors fCMA and fCMB depend on the applied field frequency, the maximum discrimination may be found by scanning the frequency empirically. Alternatively, if the dielectric property of the matter A and B can be obtained from some other methods, then the discrimination frequency may then be predicted through theoretical calculation. For example, the dielectric properties of mammalian cells may be readily determined using the technique of electrorotation in which individual cells are subjected to a rotating electrical field and cells are caused to rotate as a result of the interaction between the rotating field and the field-induced polarization. The frequency spectra of cell rotations are obtained by measuring cell rotational rate as a function of the frequency and can be analyzed in terms of dielectric shell-models to obtain cell dielectric parameters. The use of electrorational method for cell dielectric characterization is known to those skilled in the art (Wang, X.-B. et al. 1995; Huang, Y. et al, 1996; Fuhr and Hagedorn, 1996). The dielectric parameters from electrorotational measurements may then be used to calculate the frequency dependency of cell polarization factor fCM and to determine the frequency using Formula (7) at which the discrimination between two cell types is maximized.
The discrimination between matters depends also on the shape, size and configuration of the electrode elements. The change in these variables may significantly alter electrical field distribution and affect DEP forces acting on matters. Thus, it may be, necessary to design different geometries of electrode array for different applications of the present invention. Electrode array may be, for example, an interdigitated (or parallel) array, interdigitated castellated array, a polynomial array, plane electrode, or the like. Further, the array may be comprised of microelectrodes of a given size and shape, such as an interdigitated array.
In an exemplary embodiment, the signals are sinusoidal, however it is possible to use signals of any periodic or aperiodic waveform. The electrical signals may be developed in one or more electrical signal generators which may be capable of varying voltage, frequency and phase. Furthermore, DEP forces acting on matters may be programmed and varied by electrical signals applied to electrode arrays so that the signal amplitude, frequency, waveforms, and/or phases are a function of the time. For example, the applied sinusoidal signal may have a frequency (f1) for certain length of time and may then be changed to a frequency (f2). Alternatively, electrical signals with frequency-modulation (frequency continuously changes with time) and amplitude-modulation (amplitude continuously changes with time) may be applied. The signals applied to electrode arrays can therefore be programmed according to the specific separation goals and the specific separation problems. By employing such programmed signals, the DEP force may be varied with time for enhancing separation performance and the discrimination of DEP-FFF separator may be tailed to specific applications.
A chamber according to the present invention may have at least one inlet and outlet port. These ports may be the same port, or the chamber may be constructed to have different ports. The inlet port may take the form of drilled holes on the major walls of the chamber at the positions close to the chamber inlet end. The inlet port would allow the introduction of the matter to be discriminated into the chamber. The matter may be suspended or solubilized in a liquid medium, and may be introduced into the chamber through an injection valve equipped with an injection loop. The use of such injection valve for introducing the matter to be discriminated is known to those skilled in the art, as typically employed in chromatography. The inlet port may also be used for the introduction of the medium into the chamber so to establish a flow velocity profile. The reference by Wang et al (1998) provided a detailed description of using an injection valve for introducing the matter to be discriminated and for introducing the medium through an inlet port.
The outlet port may be arranged to be vertically lower than the at least one inlet port. Such an arrangement thereby permits sedimentation of the particulate matter and solubilized matter as it travels throughout the chamber. In addition to the at least one inlet port and one outlet port, the chamber may also include one or more input ducts which allow the fluid to flow through the apparatus.
The outlet port of the chamber according to the present invention may take many forms. Specifically, the outlet port may be a single port, or a plurality of ports, or an array of ports. In one embodiment,the outlet port may be two ports and may be located on the two major facing walls at positions close outlet end of a rectangular chamber. Because the matters to be discriminated attain equilibrium levitation heights in the chamber and are transported through the chamber under the influence of the flow-velocity profile, the matters may exit the chamber from one of the two outlet ports and the carrier medium may exit the chamber from the other outlet port. The advantage of this approach would increase the concentration of the matters at the port where they exit the chamber so that the matters may be collected and analyzed. In another embodiment, the matters to be discriminated may exit the two outlet ports, i.e. one population of the matter from one outlet port and all the others from the second outlet port. This embodiment may further allow the continuous operation of the discrimination and separation of the matter using the present invention. The matters may continuously be introduced into the chamber through the inlet port, and upon their introduction into the chamber, the matters would experience dielectrophoretic forces and other operational forces (such as gravity and hydrodynamic lifting forces) and would be directed towards to different levitation heights within the chamber. At these heights, all the forces acting on the matters would balance each other and the net force would be zero. During this process of moving the matters to their equilibrium positions, the flow velocity profile would carry the matter through the chamber. Depending on their levitation positions in the profile, the matter would exit the chamber at one of the two outlet ports. Which of the two ports the matter may exit from would depend on DEP and other forces acting on the matter and thus depend on the properties of the matter, allowing the discrimination of the matter.
In another embodiment, the outlet port may be located along the entire width or a part of the width of the chamber. The outlet port may be adapted to receive the matters of various shapes and sizes. For example, the size of the outlet port may vary from approximately twice the size of the matter desired to be discriminated to the entire width of the chamber. In one embodiment, the outlet port may be constructed of one or more tubing elements, such as TEFLON tubing. The tubing elements may be combined to provide an outlet port having a cross section comprised of individual tubing elements. Further, for example, the outlet port may be connected to fraction collectors or collection wells which are used to collect separated matter. As used herein, xe2x80x9cfraction collectorsxe2x80x9d and xe2x80x9ccollection wellsxe2x80x9d include storage and collection devices for discretely retaining the discriminated particulate matter and solubilized matter. Other components that may be included in the apparatus of the present invention are, for example, measurement or diagnostic equipment, such as flow cytometers, lasers, particle counters, particle impedance sensors, impedance analyzers, and spectrometers. These analytical instruments connected directly to the outlet port of the chamber may serve not only detection step for measuring and recording the time of the arrival of the particulate or solubilized matter but also analyzing step for characterizing the properties of the matter. For example, an AC impedance sensor may be connected to the outlet port of the chamber, and coupled with AC impedance sensing electronics, may serve an analytical step for determining the AC impedance of individual particles when they exit the separation chamber.
The matter being discriminated using the chamber of the present invention attains equilibrium positions within the chamber at which DEP and other forces (e.g. gravity or hydrodynamic lifting forces) balance each other. A fluid flow may be established in the chamber so to establish a flow velocity profile. After being displaced within such a fluid flow profile, the displaced matter may exit from the outlet port or ports at a time proportionate to the displacement of the matter within the fluid. Specifically, the matter equilibrated at different positions within the flow profile is carried by the fluid flow at different speeds or matter at different levels of displacement within the fluid travels at different speeds. Therefore, the matter is discriminated by its displacement within the fluid flow. Matters of different properties attain different equilibrium positions or are displaced at different levels within the fluid flow profile. Particulate matter and solubilized matter within the fluid flow velocity profile will travel through the chamber at velocities according to their positions within the velocity profile.
This velocity profile may be, for example, a hydrodynamic fluid profile such as a parabolic flow profile. For a chamber of a rectangular shape, the velocity profile may be determined by knowing the average fluid velocity, and the chamber width and thickness, as shown in Equations (1) and (2). The average fluid velocity may be calculated based on the flow rate of the fluid, and the chamber width and thickness, according to the equation:
average fluid velocity=(flow rate)/(chamber widthxc3x97chamber thickness)xe2x80x83xe2x80x83(8)
Parameters that determine the velocity profile of the fluid flow include (but are not limited to): the chamber width or thickness, which in a rectangular embodiment may be the distance between opposing walls; constrictions or expansions of the fluid flow path which may include, for example, those arising for a non-parallel disposition of opposing chamber walls, or from the presence of suitably-placed obstructions or vanes; surface roughness of the chamber walls; structural features of the chamber walls that give rise to periodic or aperiodic modifications of the thickness of the fluid stream, including the electrode elements and other surface structural configurations, and the geometrical form of the chamber which may be, for example, rectangular, circular, wedge-shaped, stepped, or the like.
In one embodiment of the present invention, an interdigitated (parallel) electrode array may be adopted on the bottom wall of a rectangular separation chamber. The matter to be discriminated is introduced into the chamber with appropriate electrical signals applied and is positioned at equilibrium heights with respect to the electrode elements under the influence of the DEP forces and the gravitational forces. Matters of different properties (e.g.: dielectric, density, size) are displaced to different heights, allowing the discrimination of the matter. Further, when a fluid flow profile such as those described in Equations (1) and (2) is generated in the chamber, the matter being displaced at different heights from the electrode plane is transported at different velocities under the influence of the fluid flow. For example, when a parabolic flow profile (along the vertical direction as in Equation (2)) is established, the matter being displaced to positions close to the half-height of the chamber travels at higher speeds than the matter being displaced to positions close to the chamber top and bottom walls. Thus the matter may be discriminated by such differing velocities. As an example, two particulate matters A and B are positioned at equilibrium heights heqA and heqB within a parabolic flow profile (along the vertical direction as in Equation (2)). Their velocities caused by the fluid flow are                                           V            A                    =                      6            ⁢                                          K                α                            ⁡                              (                                  V                  m                                )                                      ⁢                                          h                eqA                            H                        ⁢                          (                              1                -                                                      h                    eqA                                    H                                            )                                      ,                  xe2x80x83                ⁢        and                            (        9        )                                          V          B                =                  6          ⁢                                    K              α                        ⁡                          (                              V                m                            )                                ⁢                                    h              eqB                        H                    ⁢                                    (                              1                -                                                      h                    eqB                                    H                                            )                        .                                              (        10        )            
Here Kxcex1 is a factor which lies between 0 and 1, and it reflects the retardation effects due to the chamber wall (Williams et al, 1992). If the matter A is positioned higher than the matter B but less than the half chamber height H, heqB less than heqA less than H, then A would travel at larger velocity than B, VB less than VA. Further, different matter, when introduced into the chamber at a fixed time, would take differing time to travel through the chamber and to exit the chamber outlet port. The matter having larger velocities would exit the chamber ahead of the matter having smaller velocities. For the matter A and B described above, the time they take to travel through the chamber of length L would be                               t          A                =                              L                          V              A                                ⁢                      xe2x80x83                    ⁢          and                                    (        11        )                                          t          B                =                              L                          V              B                                .                                    (        12        )            
Thus, the matter may be further discriminated and separated by such differential exit-time (tB greater than tA since VB less than VA). For the matter exiting the chamber earlier may be collected separately from those exiting the chamber later, allowing the matter separation and discrimination. In another embodiment of an apparatus according to the present invention, a chamber may have two facing electrode arrays adapted on opposing surfaces. The chamber may be oriented so that the electrode planes stand substantially vertical and the thin sides of the chamber are vertically arranged. It is understood, however, that the electrode planes need not be only vertical, and the present invention contemplates adapting the apparatus at varying angles. Different electrical signals (frequency and magnitude) may be applied to the facing electrodes from the signal generator so that particles experience different cDEP forces. Further, within each electrode array, each alternate element may receive different electrical signals to create an inhomogeneous alternating electric field.
This further embodiment may have, for example, one inlet port adapted to receive the particulate matter to be discriminated. The inlet port may be located, for example, close to the top of one end of the chamber. This apparatus may also include one or more ducts to introduce a fluid that travels through the chamber. The ducts, which may be arranged substantially along the entire width of the input end of the chamber, serve to introduce a sheet of fluid that travels throughout the chamber in a substantially linear direction. As used herein, a xe2x80x9csheetxe2x80x9d of fluid may be a flow of fluid or gas entering the chamber at a substantially uniform fluid velocity. The uniform distribution in the fluid velocity here refers to that the fluid velocity does not vary with positions along the entire width of the input end of the chamber. However, the fluid velocity may be a function of the distance from electrode planes located at two major, facing walls. The introduced xe2x80x9csheetxe2x80x9d fluid carries the particulate matter through the chamber. Following transit through the chamber, fluid leaves at the opposite end. This exit end of the chamber may include, for example, one or more exit ports, which may be arranged in one or more arrays of exit ports. The outlet port may be constructed so that matter having different lateral positions at one vertical level may be separately discriminated. For example, it may be possible to utilize a laser as a tool to determine characteristics of matter exiting at selected lateral positions.
Different electrical signals (frequency or magnitude or both) are applied to electrode elements located on each of the side walls. There is a synergistic interaction between these different electrical signals which creates an inhomogeneous electric field. Particulate matter to be discriminated is subjected to DEP forces (FDEP1 and FDEP2) from electrical fields induced from electrode elements located on both the side walls,
Ftotal=FDEP1+FDEP2.xe2x80x83xe2x80x83(13)
Under the combined influence of these forces, the matter is directed equilibrium positions with respect to the side-walls. The equilibrium position, defined as the distances from the two side-walls, is therefore determined by DEP forces on the particulate matter. Since both DEP force components are dependent on the dielectric and conductive properties of the matter, this equilibration position depends on these properties of the matter. Other factors influencing the equilibrium positions include the magnitude and frequency of the electrical fields applied to the electrodes on the opposing chamber walls, the fluid density, viscosity, and flow rate. Different matter, because of their different properties, equilibrates at different characteristic distances from the side-walls of the chamber and attains different equilibrium positions based on this synergistic interaction of the DEP forces induced by the differing electrical signals. When a fluid flow is established in the chamber, the velocity of the different matter within the fluid is controlled by the velocity profile of the fluid and the equilibrium position of the matter with in the flow-velocity profile. This velocity profile has a maximum velocity towards the center of the chamber, with this velocity proportionately diminishing as distance from the side-walls decreases. Because of this velocity profile, matter that has equilibrated at different equilibrium distances from the chamber walls will be carried at different velocities and therefore take varying amounts of time to traverse the chamber. Those skilled in the art would appreciate that the equations describing the velocity and the transit time of matter through the chamber under the influence of the fluid flow are similar to Equations (9, 10, 11, 12).
The distance that matter sediments during its passage across the chamber will depend upon its transit time, as gravitational forces act on the matter during its transit through the chamber, and this is known as a xe2x80x9csedimentation effect.xe2x80x9d For a spherical particle having a radius r and density xcfx81p, the sedimentation velocity Vsed in a medium having a density xcfx81m and viscosity xcex7m can be written as                               V          sed                =                                            2              ⁢                              xe2x80x83                            ⁢                              r                2                            ⁢                              xe2x80x83                            ⁢                              (                                                      ρ                    p                                    -                                      ρ                    m                                                  )                                                    9              ⁢                              xe2x80x83                            ⁢                              η                m                                              .                                    (        14        )            
The sedimentation velocity is a function of the particle size and density, medium viscosity and density. Consequently, different particles will sediment to different depths (Dsed) based upon the sedimentation velocity (Vsed) and the transit time (ttransit) of matter through the chamber,
Dsed=Vsedxc3x97ttransit.xe2x80x83xe2x80x83(15)
Thus, particle sedimentation depends on matter characteristics, such as size, mass, and volume, for example. As described above, the time (ttransit) required for particles to travel across the entire length of the chamber is controlled by the fluid flow profile and the positions of particles within the flow-velocity profile. The placement of particles within the fluid flow profile is in turn determined by the synergism of the differing electrical signals. Thus, particles with different characteristics (e.g.: dielectric property, size) may be placed at different positions in the flow profile and therefore exhibit different transit times. The combination of differences in transit time and in sedimentation velocity between particles of different properties (e.g. dielectric property, density, size) may lead to different sedimentation depths for these particles. They may exit the chamber through different outlet ports which may be placed at different heights with respect to the inlet ports. Discrimination may be accomplished either in xe2x80x9cbatch modexe2x80x9d or in xe2x80x9ccontinuous mode.xe2x80x9d In batch mode, an aliquot of particles is injected and collected with respect to the time of transit (ttransit) for the particles and the height of exit (Dsed) at the outlet ports. In continuous mode, a constant stream of particles is injected into the inlet port, and matter emerging at different heights (Dsed) are continuously collected.
The methods and apparatus of the present invention introduce for the first time the use of the frequency-dependent dielectric and conductive properties of particles as well as those of the suspending medium. These new criteria for particle fractionation allow sensitive manipulation of particles because the dielectrophoretic force is large and strongly dependent on particle properties. Appropriate choices of the suspending medium and applied field conditions allow for high levels of discrimination.
Previously reported field flow fractionation techniques have limitations for biological samples because of the narrow range of cell densities, demanding complex centrifuges and centrifugation techniques for good discrimination. The cDEP affinity method demands large differences in the dielectric characteristics of the particles to be separated so that selected particulate matter and solubilized matter can be completely immobilized while others are swept away by fluid flow forces. Since, for biological cells, damage can occur at high electric field strengths, there is a practical limitation to the maximum cDEP force that can be applied and this in turn limits the maximum fluid flow rate in the cDEP affinity approach. This may result in a slow cell sorting-rate. In the methods of the present invention, these limitations are substantially reduced. Furthermore, the cDEP affinity method of the prior art utilizes the dielectrophoretic force component that generally immobilizes particles on electrode elements. The cDEP/FFF approach of the present invention utilizes the DEP forces and other forces to determine the positions of particles or other matter in a flow-velocity profile and exploits the flow-velocity profile.
Also, in the present invention, the flow profile is an active mechanism for the separation and discrimination of particles, and the dielectrophoretic force (mainly the force component in the direction normal to the fluid flow direction), in conjunction with other forces (e.g. gravity, hydrodynamic lifting force, or another dielectrophoretic force), is the primary means by which the heights or positions of particles in the fluid flow profile are controlled. As discussed above, the fluid profile may be controlled by apparatus design, fluid rate, density and the like. By combining FFF and dielectrophoretic forces, the present invention takes advantage of particle volume and density in synergism with the frequency-dependent particle dielectric and conductive properties as well as surface configuration. The operation of an apparatus according to the present invention may be controlled by varying experimental conditions including, but not limited to, the particle suspending medium conductivity and permittivity, the fluid flow rate, viscosity and density, the applied electrical field strength, the applied frequency and the applied electrical signal waveform. This utilization of many parameters in setting the operational conditions for fractionation greatly increases the ability to discriminate between different particulate matter and solubilized matter. In the methods according to the present invention, particles emerging from the outlet ports of the apparatus may be collected, for example, by one or more fraction collectors, or may be fed directly into analytical apparatus such as flow cytometer or impedance sensors to characterize separated particles. Furthermore, when necessary or desired, particles may be transferred to collection wells containing appropriate solutions or media, such as neutral salt buffers, tissue culture media, sucrose solutions, lysing buffers, solvents, fixatives and the like. In the case of biological cells, the collected, separated cells may be further cultured and analyzed for their molecular characteristics. Alternatively, the separated cells may be subjected to other molecular, biochemical studies.
In an illustrative embodiment, the chamber may be constructed in a rectangular shape using, for example, two glass slides as chamber walls. These chamber walls may be spaced apart by spacers to create the rectangular design. These spacers may be made of, for example, glass, polymeric material such as TEFLON, or any other suitable material. The size of the chamber and spacing between chamber walls is dependent on the size of the particles which are to be discriminated. To practice the methods of the present invention, an apparatus may have spacing between about 100 nm and about 10 mm, and more preferably between about 20 microns and about 600 microns in an illustrative embodiment for the purpose of discriminating mammalian cells. Further, a longer chamber may be desired to permit greater discrimination throughput. An apparatus according to the present invention can discriminate cells at a rate between about 100 and about 3 million cells per second. Factors that determine discrimination rate include, for example, the dielectric properties of the particles to be discriminated, the electrode design, length of the chamber, fluid flow rate, frequency and voltage of the electrical signals, and the signal waveforms. The chamber dimensions may be chosen to be appropriate for the input matter type, characteristics, and degree of discrimination desired or required.
In other embodiments, one or more surfaces of the chamber may support an electrode array. The electrode array may be a microelectrode array of, for example, parallel electrode (interdigitated) elements. In certain embodiments, the parallel electrode elements may be spaced about 20 microns apart. The apparatus may accommodate electrode element widths of between about 0.1 microns and about 1000 microns, and more preferably between about 1 micron and about 100 microns for embodiments for the discrimination of cellular matter. Further, electrode element spacing may be between about 0.1 microns and about 1000 microns, and for cellular discrimination more preferably between about 1 micron and about 100 microns. Alteration of the ratio of electrode width to electrode spacing in the parallel electrode design changes the magnitude of the dielectrophoretic force and thereby changes the particle levitation characteristics of the design. The electrode elements may be connected to a common electrical conductor, which may be a single electrode bus carrying an electrical signal from the signal generator to the electrode elements. Alternately, electrical signals may be applied by more than one bus which provides the same or different electrical signals. In certain embodiments, alternate electrode elements may be connected to different electrode buses along the two opposite long edges of the electrode array. In this configuration, alternate electrode elements are capable of delivering signals of different characteristics. As used herein, xe2x80x9calternate electrode elementsxe2x80x9d may include every other element of an array, or another such repeating selection of elements. The electrode elements may be fabricated using standard microlithography techniques that are well known in the art. For example, the electrode array may be fabricated by ion beam lithography, ion beam etching, laser ablation, printing, or electrodeposition. The array may be comprised of for example, a 100 nm gold layer over a seed layer of 10 nm chromium or titanium. An apparatus according to the present invention may be used with various methods of the present invention. For example, an apparatus according to the present invention may be used in a method of discriminating particulate matter and solubilized matter utilizing dielectrophoresis and field flow fractionation. This method includes the following steps.
First, the chamber is preloaded through one inlet port with a carrier medium, such as a cell suspension medium, tissue culture medium, a sucrose solution, or the like. Cautions should be taken during the loading to ensure that no bubble (or only few small bubbles) is introduced into the chamber.
Secondly, the matter to be discriminated suspended or solubilized in a medium may then be introduced into one or more inlet ports of the chamber. During this introduction, certain electrical signals may be applied so that the matter to be discriminated is subjected to dielectrophoretic forces which may prevent the matter to be in contact with the walls containing the electrode elements. Alternatively, in some applications, no electrical signals are applied during the introduction of the matter.
Thirdly, after the introduction of the matter into the inlet region of the chamber, certain electrical signals may be applied for some time prior to the commencement of the fluid flow in the chamber. During this period, the matter may move to their appropriate positions (or equilibrium positions) under the influence of dielectrophoretic forces generated by the application of electrical signals and other forces such as gravity. At these positions, all the forces acting on the matter balance each other and the net force is zero or close to zero. The matters of different characteristics may attain different equilibrium positions within the chamber and are therefore discriminated according to their equilibrium positions. Alternatively, in some applications, no electrical signals are applied so that the matter may move to appropriate positions (equilibrium positions) under the influence of forces such as gravity.
Finally, a fluid flow is established in the chamber by, for example, pumping the carrier medium into the chamber using a syringe pump. This causes the carrier medium to travel through the chamber according to a velocity profile so that the velocity of the medium at different positions with respect to the chamber walls may be different. At least one alternating electrical signal may be applied to the one or more electrode elements, which creates an inhomogeneous alternating electric field within the chamber. This field causes dielectrophoretic forces to act on the matter within the chamber. Dielectrophoretic forces, together with other forces such gravity and hydrodynamic lifting forces, cause the matter to be displaced to equilibrium positions in the flow velocity profile within the carrier medium. At these equilibrium positions, the dielectrophoretic forces are balanced by other forces acting on the matter. The matters are discriminated according to their positions within the carrier medium. In addition, the matters at different positions are caused to travel at different velocities under the influence of the flow-velocity profile of the carrier medium. Thus, the matters are further discriminated according to their velocities. To further discriminate matter, the frequency, or magnitude or both of the electrical signal may be varied with time. Such change thereby causes a change in the inhomogeneous alternating electric field which, in turn, changes the dielectrophoretic forces acting on the matter and alters the displacement of the matter with respect to the electrode elements. These changes further influence the equilibrium positions of the matter within the flow velocity profile and the velocity of the matter. The matters are further discriminated according to their exit time from the chamber. The matter having larger velocities will exit the chamber ahead of others having small velocities. The matter after exiting the chamber may be collected and/or analyzed.
Another method according to the present invention for discriminating particulate matter and solubilized matter using dielectrophoresis and field flow fractionation includes the following steps. First, the chamber is preloaded through one inlet port with a carrier medium, such as a cell suspension medium, tissue culture medium, a sucrose solution, or the like. Secondly, the matter to be discriminated suspended or solubilized in a medium may then be flown into one or more inlet ports of the chamber. This introduction causes the carrier medium to travel through the chamber according to a velocity profile. The carrier medium at different positions of the chamber may travel at different velocities. At least one alternating electrical signal may be applied to the one or more electrode elements, which creates an inhomogeneous alternating electric field within the chamber. This field generates dielectrophoretic forces acting on the matter and causes the matter within the chamber to be displaced to a position in the flow velocity profile within the carrier medium. At such an equilibrium position, dielectrophoretic force acting on the matter is balanced by other forces such as gravity, or hydrodynamic lifting forces, or another dielectrophoretic force in the chamber. Thus, the matter is discriminated according to its position within the carrier medium. Furthermore, the matter may be discriminated, for example, according to its velocity. The matter at different positions of the flow velocity profile travels at different velocities under the influence of the fluid flow. To further discriminate matter, the electrical signal may be varied (frequency, or magnitude, or both). Such a change thereby causes a change in the inhomogeneous alternating electric field which, in turn, changes the dielectrophoretic force acting on the matter and changes the displacement of the matter with respect to the electrode elements. These changes further influence the equilibrium positions of the matter within the flow velocity profile and the velocity of the matter. The matters are further discriminated according to their exit time from the chamber. The matter having larger velocity will exit the chamber earlier than other having smaller velocities. The matter after exiting the chamber may be collected and/or analyzed.
Another method according to the present invention includes discriminating particulate matter and solubilized matter utilizing dielectrophoresis and field flow fractionation according to the following steps. First, the chamber is preloaded through one inlet port with a carrier medium, such as a cell suspension medium, tissue culture medium, a sucrose solution, or the like. Secondly, the matter to be discriminated is introduced into the chamber through one inlet port of a chamber. Next, a transport fluid, which may be, for example, a tissue culture medium or a gas, is flown into at least one duct. This causes a fluid flow in the chamber according to a velocity profile. The fluid at different positions of the chamber may travel at different speeds. At least one electrical signal is applied to at least one electrode element. These one or more electrical signals thereby create an inhomogeneous electric field within the chamber. The field causes a DEP force on the matter causing the matter to be displaced to a position within the transport fluid. At such an equilibrium position, DEP force is balanced by other forces (such as gravity, hydrodynamic lifting forces or another DEP force) acting on the matter. The matter is discriminated according to its position in the flow velocity profile within the transport fluid. As this transport fluid is subjected to a velocity profile, the matter moving at different velocities is thereby partitioned according to its position in the direction of the fluid flow. The matter is discriminated according to its velocity and its position within the fluid flow. To further discriminate matter, the electrical signal may be varied (frequency, or magnitude, or both). Such a change thereby causes a change in the inhomogeneous alternating electric field which, in turn, changes the dielectrophoretic force acting on the matter and changes the displacement of the matter with respect to the electrode elements. These changes further influence the equilibrium positions of the matter within the flow profile and the velocity of the matter. Furthermore, the separated particulate or solubilized matters may be collected at times dependent upon their velocities. It is further possible to collect the matter at one or more outlet ports for further analysis and characterization.
Another method according to the present invention for discriminating particulate matter and solubilized matter utilizing dielectrophoresis and field flow-fractionation includes the following steps. In this case, a continuous discrimination and separation of particulate matter and solubilized matter is achieved. The chamber according to the present invention has two outlet ports located on the two major facing walls. At least one of the two major walls supports an electrode array. First, the chamber is preloaded through one inlet port with a carrier medium, such as a cell suspension medium, tissue culture medium, a sucrose solution, or the like. Secondly, the matter to be discriminated suspended or solubilized in a medium is continuously introduced into the chamber. This causes a fluid flow in the chamber according to a velocity profile. At least one electrical signal is applied to at least one electrode element. These one or more electrical signals thereby create an inhomogeneous electric field within the chamber. The field causes a DEP force on the matter causing the matter to move towards equilibrium position within the transport fluid. Thus, the matter is not only carried with the fluid flow but also driven by combined influences of DEP and other forces such as gravity, hydrodynamic lifting forces or another DEP forces. When the matter reaches the outlet end, it will exit the chamber at one of the two outlet ports, depending on its position along the direction normal to the two major walls of the chamber. The matter is discriminated according to the outlet port it exits the chamber. For example, if the matter consists of two subpopulations having different dielectric properties, the two subpopulation of the matter may attain different positions in the fluid flow when they reach the chamber outlet end. The separation of the two populations is achieved since the two populations exit the chamber at the two different outlet ports. Clearly the discrimination of the matter using this approach can be operated continuously. The separated matter from the two outlet ports may be further collected and analyzed.
There are further steps possible to more precisely discriminate matter. These steps include the following. First, the alternating electrical signal or signals may be selected at a frequency and voltage combination which causes the matter to be either attracted towards or repelled from the electrode elements. By doing so, the matter is more clearly displaced within the transport fluid. By application of such a voltage and frequency combination, it is possible to hold the matter in close proximity to the electrode elements.
It is possible to select a frequency to attract desired or nondesired matter. As used herein, desired matter may be any matter which is desired to be discriminated and collected for further use. For example, the separation of normal blood cells from a sample containing xe2x80x9ccontaminatedxe2x80x9d cancer cells may be desired for use in returning these normal cells into a patient""s bloodstream. So normal cells may be called xe2x80x9cdesired matterxe2x80x9d in this case. Nondesired matter may be matter which is desired to be discriminated for other purposes. For example, cancer cells from a patient""s blood or bone marrow may be discriminated so that a sample of blood not containing the cancer cells may be returned to the patient. In this case, the cancer cells may be called xe2x80x9cnondesired matterxe2x80x9d.
A method for discriminating such a combination of matter may include the following. A frequency is selected so that the nondesired matter is held in close proximity to the electrode elements while simultaneously the desired matter is carried with the fluid flow and is separated from the nondesired matter. This frequency may be known as a holding frequency. The fluid flow then carries the desired matter to the outlet port or ports of the chamber, where it may be collected. During this process, the desired matter may also be subjected to further discrimination and separation so that the subpopulations of the desired matter are separated under the cDEP/FFF operation. After collection, the desired matter may, for example, be returned to a patient""s bloodstream or bones, or it may be used for diagnosis or other molecular or biochemical analysis. Then, to clear the chamber, the frequency may be changed, or the voltage itself may be turned off. This will cause the nondesired matter to be released from close proximity to the electrode element and will be partitioned by the fluid flow. This nondesired matter may then flow through the chamber in the fluid, and may be collected, if required. After collection, the nondesired matter may be used, for example, for diagnosis or other purposes.
In an alternate embodiment, it may be possible to hold desired matter in close proximity to the electrode elements, and first partition the nondesired matter by the fluid flow, following the same steps outlined above.
The apparatus and methods of the present invention may be used for a number of different useful manners. For example, the methods according to the present invention may be used to determine characteristics of an unknown particulate matter and unknown solubilized matter in a sample of matter. These characteristics can then be compared to known matter. Additionally, the methods of the present invention may be used to diagnose a condition by determining a presence of unidentified particulate matter and unidentified solubilized matter in a patient sample. This unidentified matter may be, for example, the presence of a cancer, a virus, parasite, or the like. After determining the presence of a condition, the methods of the present invention may be used to treat the condition by using an apparatus according to the present invention to discriminate the cancer, virus, parasite or the like from normal blood or bone marrow cells.
xe2x80x9cManipulation or discriminationxe2x80x9d as used in relation to the present invention may include, for example, characterization, separation, fractionation, concentration and/or isolation.
Typical biological applications for the device useful for specific products and services include the manipulation or discrimination of tumor cells, such as epithelial tumor cells or leukemia cells, from blood and hemopoietic stem cells, purging of tumor cells from bone marrow and hemopoietic stem cells and mixtures with other normal cells, purging of residual T-lymphocytes from stem cells, and enrichment of specific target cell types including tumor cells, stem cells, etc. Also included is the manipulation or discrimination of leukocyte cell subpopulations, removal and concentration of parasitized erythrocytes from normal erythrocytes in malaria and of other parasitized cells from their normal counterparts, manipulation of cells at different phases of the cell cycle, manipulation of viable and non-viable cells, manipulation of free cell nuclei, and manipulation of nucleated fetal erythrocytes and trophoblast cells from maternal blood for further analysis including genetic testing. Moreover, the invention contemplates the manipulation of bacteria, viruses, plasmids and other primitive organisms from water, blood, urine, cell mixtures and other suspensions, manipulation and identification of tumor cells in biopsies, plaques and scrape tests including Pap smears, and the manipulation and identification of metastatic tumor cells from cell mixtures.
With different and smaller electrode geometries, it is contemplated that the technology can be used for molecular applications including manipulation of DNA or RNA molecules and/or DNA or RNA fragments according to their molecular weight, folding characteristics and dielectric properties, manipulation of chromosomes, manipulation of specific protein/DNA and protein/RNA aggregates, manipulation of individual proteins from a mixture, and manipulation of specific subcellular molecular complexes and structures.
The chamber used for cDEP/FFF application may vary significantly in size to fit the need of different sample sizes. For example, the large size chamber may be implemented for separating many millions of the cells at each operation. On the other extreme, the chamber may be miniaturized so to form a microfluidic cell separation step in an integrated bioanalytical system. Such miniaturized chamber may be integrated with other microfluidic devices or components. In order to optimize particle discrimination in different applications it is understood that the present invention may encompass use of specifically-targeted electrodes and chamber designs. These designs should provide a sensitive dependency of the height of particle levitation on the particle dielectric properties. For example, alteration of the ratio of electrode width to electrode spacing in the parallel electrode design changes the vertical component of the dielectrophoretic force and thereby changes the particle levitation characteristics of the design. Other strategies for providing improved particle discrimination include, for example, using more than two sets of electrode elements with different frequencies and/or voltages applied to them and the exploitation of synergism between electrical signals applied to electrode arrays on both the chamber bottom and top walls. In addition, dielectric (i.e. non-conducting) elements can be placed within the chamber to modify both the electrical field distribution and the hydrodynamic flow profile. The electrode element size and shape may be designed to optimize discrimination. Furthermore, several electrode geometries (energized with the same or different electrical signals) can be connected serially so as to provide for stepwise discrimination between different particulate matter and solubilized matter. Different chamber configurations can also be used in series. Finally, cells that have been separated by an upstream cDEP/FFF configuration can be collected and held downstream by cDEP trapping for characterization.